Cation exchange chromatography (CEX) has been effectively used as an intermediate or polishing step to remove aggregate impurities in typical mAb purification processes. The typical step is developed by binding the mAb at pH 4-6 and identifying the elution condition through salt gradient experiments at the same binding pH. However, novel biological molecules such as bispecific antibodies (BsAb) may have different structural characteristics that could result in similar charge profiles between the aggregates and the monomer. The similarity between the charge profiles may render the traditional mAb CEX step development approach ineffective.<br/>We report a BsAb case study where CEX step process development was performed to separate dimer and HMW aggregate species from the monomer. Dimer of this specific BsAb exhibited a very similar charge profile to the monomer according to analytical weak cation exchange chromatography analysis. The similarity in charge profile posed a difficult purification challenge to achieve at preparative scale chromatography with step elution. Using the traditional mAb step development approach of salt gradient experiments at pH 5-6 resulted in poor resolution between the monomer and dimer. In order to separate the monomer and dimer a unique approach of a simultaneous step change in pH and salt for elution was explored. Two different buffer systems, Phosphate-Acetate and Tris-Acetate provided the same degree of separation and yield. The Tris-Acetate system was selected for better process linkage with the downstream Anion Exchange polishing step. With the Tris-Acetate system, high aggregate removal was achieved (&gt;10%) while maintaining high monomer yield (&gt;90%).<br/>In order to better understand and control the unit operation, QbD methodology was applied that examined the effect of interaction of input parameters such as load challenge and temperature had on the separation. Finally, data from a surface response DOE was utilized to model the unit operation in order to better predict the behavior of the separation at bench and pilot scale.

Development of challenging aggregate removal step in a bi-specific antibody purification process: A case study

251st National Meeting (2016)

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